Neutrophil mobilization to inflammatory sites is a multi-step process which involves the reversible adhesion to specific endothelial plasma membrane molecules, upregulation of selected neutrophil adhesion molecules during chemoattractant exposure, and migration (chemotaxis) through endothelial gaps and the basement membrane into tissues. The present study was undertaken to examine the changes that occur in adhesion molecular expression during neutrophil chemotaxis in vitro on a polycarbonate membrane substratum. The polycarbonate membrane system was selected for this study because: 1) unlike endothelial monolayers which have dynamic changes in their adhesion molecular expression, the polycarbonate surface remains relatively constant during neutrophil adherence and activation, and 2) two populations of neutrophils are easily identified and separated with the polycarbonate membrane system; one population does not migrate to chemoattractant and remains on the upper surface of the membrane, whereas the other population migrates through the membrane pores to the lower surface of the membrane (chemotactically responsive population). Neutrophils, incubated in suspension with or without the N-formyl peptide (FMLP) for 35 minutes, were compared with the chemotactically responsive and the nonresponsive subpopulations which were exposed to a gradient of FMLP for 35 minutes. Neutrophils were stained with a panel of adhesion molecule monoclonal antibodies which recognize: the leukocyte-cell adhesion molecular family (Leu-CAM), CD11a, CD11b, CD11c, and CD18; the platelet/endothelial cell adhesion molecule-1 (PECAM-1), CD31; leukosialin, CD43; and, the homing receptor Pgp-1, CD44. Flow cytometric analysis of the panel revealed that the chemotactically responsive subpopulation consistently down-regulated the expression of these adhesion molecules. CD44, CD43, and CD11a were the most dramatically down-regulated; CD11c, CD31, and CD11b were moderately down-regulated; and CD18 was slightly down- regulated. In contrast, only CD43 was consistently down-regulated on nonadherent, FMLP-stimulated neutrophils.